1. Field of the Invention
The invention pertains to the field of baculovirus expression. More particularly, the invention pertains to novel cell lines resistant to apoptosis and nutrient stresses.
2. Description of Related Art
The baculovirus expression vector system is one of the most effective and widely used eukaryotic protein expression systems available. In the baculovirus expression system, foreign genes are inserted into the baculovirus genome and are typically expressed under the transcriptional control of one of the hyper-expressed baculovirus very late promoters, polyhedrin or p10. In addition to very high-level protein production, an additional advantage of the baculovirus expression system is the quality of post-translational processing of proteins. Foreign proteins expressed in baculovirus-infected insect cells are post-translationally processed in a manner very similar if not identical to that observed for proteins translated in other higher eukaryotes, particularly vertebrates. The baculovirus system is especially useful for production of secreted and membrane-bound proteins from higher eukaryotes since protein folding and processing in the baculovirus system is typically similar to other higher eukaryotes and usually results in biologically active proteins. However, some post-translational processes, such as trimming and modification of complex carbohydrates, differ from those observed in vertebrate cells.
Protein production in this expression system occurs in the context of a lethal infection. Thus, the physiological state of the host cell during infection may influence the level of recombinant protein translation, protein processing, and protein trafficking or secretion. Factors which influence host cell health or viability could influence protein production and quality, as well as the overall utility of this expression system. The health, growth, and propagation of host cells prior to infection by recombinant baculoviruses is also an important practical concern for large scale production of proteins in the baculovirus system.
An integral component of baculovirus expression technology is the insect cell line in which recombinant proteins are expressed. Desirable characteristics for such cell lines include the capacity to scale cultures to high cell densities, cell growth in serum free medium, growth in suspension cultures, and high level protein expression. Two cell lines, Sf9 and Sf21, are routinely used since these cells grow to high density, express reasonably high levels of protein, adapt well to serum-free media formulations, and are easily adapted to large scale suspension cultures. Sf9 cells are a derivative of the Sf21 line.
It has been demonstrated that Autographa californica nucleopolyhedrovirus (AcMNPV) infection induces apoptosis in Sf21 cells if the AcMNPV p35 gene is inactivated or absent (Clem, R. J. et al., 1991, Hershberger, P. A. et al., 1994). Numerous subsequent studies further characterized the role and function of the P35 protein as a potent general suppressor of apoptosis (Rabizadeh, S. et al., 1993, Sugimoto, A. et al., 1994, Hay, B. A. et al., 1994, Beidler, D. R. et al., 1995, Martinou, I. et al., 1995, Robertson, N. M. et al., 1997).
The P35 protein inhibits programmed cell death by inhibiting cellular caspases, a family of cysteinyl proteases that are important components or effectors of the cell death pathway (Martin, S. J. and Green, D. R., 1995, Cryns, V. and Yuan, J., 1998). P35 has been shown to inhibit caspases belonging to groups I, II, and III and inhibition is believed to result from a multistep mechanism. P35 interacts directly with cellular caspases and is cleaved (at aspartate 87) by the caspase (Xue, D. and Horvitz, H. R., 1995, Bertin, J. et al., 1996, Bump, N. J. et al., 1995, Fisher, A. J. et al., 1999).
Cleavage of P35 is believed to result in a conformational change that is required for P35 inhibition of the caspase. Thus, P35 appears to represent an irreversible inhibitor of caspases since P35 fragments of 10 and 25 kDa remain associated with the caspase after cleavage at P35 residue Asp87 (Bump, N. J. et al., 1995, Fisher, A. J. et al., 1999).
However, P35 cleavage alone does not appear to be sufficient for inhibition of caspase activity since a single amino acid substitution mutation in P35 prevents the stable association of P35 with the caspase (but not its cleavage), and P35 containing this mutation does not inhibit caspase-3 activity (Fisher, A. J., et al. 1999).
A previous study showed that the P35 protein expressed in stable cell lines was capable of inhibiting apoptosis induced by actinomycin D or by a mutant virus in which the p35 gene was deleted (Cartier, J. L., et al. 1994). In that study, stably transfected cells expressing p35 did not appear to have increased viability after infection, when compared with untransfected Sf21 cells. Resistance of those p35 expressing cells to nutrient stress was not examined, nor was the expression of secreted proteins from baculovirus expression vectors examined.
A mammalian oncogene, bc1-2, is normally expressed in the T and B-lymphocytes. It is believed that the product of this gene is a “survival gene” normally involved in enhancing cellular survival. Bc1-2 has been stably transfected into an immortalized cell line (Milner et al., 1992). The stable transfectants were selected using G418.
Another study utilized these bc1-2 expressing cell lines to test for survival of these cells under apoptotic and high stress conditions (Singh et al., 1996). The growth characteristics of the bc1-2 overexpressing cell lines were compared with control cells in stationary, suspension and serum-free cultures. In each of these cases, bc1-2 suppressed apoptosis and the cells were more robust than the control cells. This study concentrated on overexpressing bc1-2, a mammalian protein that is otherwise normally expressed in the cells. No viral genes encoding suppressors of apoptosis were incorporated into the cell lines. Bc1-2 is a suppressor of apoptosis that is expressed normally from mammalian cells and does not block apoptosis as broadly as certain viral suppressors of apoptosis, such as baculovirus P35, Cowpox CrmA or Vaccinia SPI-2. Thus, the use of viral suppressors of apoptosis may provide substantial advantages over cellular proteins. In the former study of bc1-2 expressing cells, only cell growth and responses to modified nutrient regimes were examined. They did not examine the effects of apoptotic suppression on foreign gene expression or on protein production.
Spodoptera frugiperda Sf9 cells provide desirable growth characteristics and are extensively used in both industrial and research applications. In general, recombinant protein expression in insect cells infected with baculovirus expression vectors is much higher than in mammalian cells infected with vertebrate virus expression vectors. Sf9 cells, as well as most insect cell lines, are limited by their susceptibility to stress which results in the induction of apoptosis. Another widely used cell line derived from Trichoplusia ni (BTI-Tn5B1-4 or HighFive™ cells) provides greatly improved protein expression but is more difficult to grow in suspension cultures, and may not be suitable for some types of scale-up applications. Sf9 cells do not generate the same high levels of protein expression that can be achieved from Tn5B1-4 cells. Thus, neither of these cell lines provides the optimal combination of features for high-level protein production.